Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing

Abstract

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing approximately 70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.

Figure 1.—

Coverage and quality analysis of the third chromosome from A15 and 791 runs. (A) Distribution of nucleotide coverage depth for the original A15 third chromosome and for the 791 mutagenized third chromosome. The heat map indicates pairwise coverage. (B) Distribution of MAQ consensus nucleotide quality scores for A15 and 791 for nucleotides of the third chromosome. Scores are shown only for consensus nucleotides that were not ambiguous and had a depth of at least 4. Heat map indicates pairwise quality.

Figure 2.—

Analysis of SNPs between the original A15 and the mutagenized 791 chromosomes. (A) Classification, verification, and confirmation information for initial set of 165 candidate SNPs. (B) Gene conversion clusters. For each SNP cluster, the 3RT nucleotide is shown above, the A15 is shown in the middle, and the 791 nucleotide is shown below. Yellow indicates identity with the 3RT nucleotide, and red indicates a nucleotide that is different from the 3RT nucleotide. The relevant balancer sequence is shown to the right of each cluster, with the inferred gene conversion event indicated by a red arrow. Relative spacing of SNPs is shown with a scale bar. (C) Distribution of verified variants along the third chromosome, EMS canonical G/C-to-A/T differences above, and noncanonical EMS differences below. Gene conversion clusters of mutations are indicated by red stars.

Figure 3.—

Annotation of encore. (A) A C-to-T transition turns the 1353 glutamine codon to a premature stop. (B) Complementation test of encore⁷⁹¹ lesion. Embryos of mothers raised at 18° were assayed for the fused dorsal appendage phenotype. 3RT indicates the target chromosome from which the A15 chromosome was derived.